In plants, disease resistance to fungal, bacterial and viral pathogens is associated with a plant response termed the hypersensitivity response (HR). In the HR, the site in the plant where the potential phytopathogen invades undergoes localized cell death, and it is postulated that this localized plant cell death aspect of the HR contains the invading microorganism or virus, thereby protecting the remainder of the plant. Other plant defenses include the production of phytoalexins (antibiotics) and/or lytic enzymes capable of averting pathogen ingress and/or cell wall modifications which strengthen the plant cell wall against physical and/or enzymatic attack.
The HR of plants, including tobacco, can include phytoalexin production as part of the response to invading microorganisms. One class of compounds made boy tobacco (Nicotiana tabacum) in response to microbial invaders are the sesquiterpenes.
Cell suspension cultures have provided useful information regarding the regulation of terpene synthesis. Isoprenoids are ubiquitous in nature, and the early portions of the biosynthetic pathway are shared with the biosynthetic pathway for other isoprenoid compounds such as sterols, carotenoids, dolichol and ubiquinone and growth regulators (e.g., gibberellic acid), which are classified as primary metabolites. Isoprenoid compounds classified as secondary metabolites are not essential for growth, and include mono-, sesqui- and diterpenoids. These secondary metabolite isoprenoids are important mediators of interactions between the plant and its environment.
A variety of compositions can serve as elicitors of plant phytoalexin synthesis. These include, but are not limited to, one or more toxic ions, e.g., mercuric ions, other chemically defined compositions, metabolic inhibitors, cell wall glycans, certain glycoproteins, certain enzymes, fungal spores, chitosans, certain fatty acids and certain oligosaccharides derived from plant cell walls [See, e.g., Sequeira, L. (1983) Annu. Rev. Microbiol. 37:51-79 and references cited therein]. Epi-5-aristolochene synthase (EAS) activity in tobacco plants has been shown to be induced by cell wall fragments of certain Phytophthora species and by Trichoderma reesei cellulase but not Aspergillus japonicum pectolyase [Chappell et al. (1991) Plant Physiol. 97:693-698]. Attack by other plant pathogens or an avirulent related strain can also induce phytoalexin synthesis; for example, Pseudomonas lachrymans induces sesquiterpenoid synthesis in tobacco [Guedes et al. (1982) Phytochemistry 21:2987-2988].
Elicitins are proteins which are produced by plant pathogens and potential plant pathogens, which proteins induce the HR in tobacco plants. Amino acid and nucleotide coding sequences for an elicitin of Phytophthora parasitica have been published [Kamoun et al. (1993) Mol. Plant-Microbe Interactions 6:573-581]. Plant pathogenic viruses including, but not limited to, Tobacco Mosaic Virus (TMV) induce the HR in infected plants. Bacteria which infect plants also can induce HR and thereby disease resistance; representative bacteria eliciting HR include, but are not limited to, Agrobacterium species, Xanthomonas species and Pseudomonas syringae. Plant pathogenic fungi (and certain avirulent strains as well) also induce the HR response, where these include, but are not limited to, Phytophthora parasitica and Peronospora tabaci.
When tobacco cell suspension cultures are treated with an elicitor, squalene synthetase is suppressed, thus stopping the flow of common biosynthetic precursors into sterols. The concomitant induction of sesquiterpene cyclase gene expression causes the flow of precursors into sesquiterpenes. The first step in the pathway from farnesyl diphosphate to the sesquiterpene phytoalexin capsidiol in elicitor-induced tobacco tissue is catalyzed by 5-epi-aristolochene synthase (EAS), a sesquiterpene cyclase. The coding sequence and deduced amino acid sequence for one member of the EAS gene family of tobacco have been published [Facchini and Chappell (1992) Proc. Natl. Acad. Sci. USA 89:11088-11092]. The transcriptional expression of one or more members of the EAS gene family is induced in response to elicitors.
There is a long felt need in the art for methods of protecting plants, particularly crop plants, from infection by plant pathogens, including but not limited to, phytopathogenic viruses, fungi and/or bacteria. Especially important from the standpoint of economics and environmental concerns are biological or "natural" methods rather than those which depend on the application of chemicals to crop plants. There is also a long felt need in the art for plant transcriptional regulatory sequences for use in controlling the expression of heterologous DNA sequences in transgenic plants.